This application addresses broad Challenge Area (08) Genomics and specific Challenge Topic, 08- MH-103: Understanding the genomic risk architecture of mental disorders. Rett syndrome (RTT), caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MeCP2), is arguably the best characterized of the autism spectrum neurodevelopmental disorders. RTT is hypothesized to result from inappropriate neuronal maturation, altered synaptic connectivity and plasticity, possibly through abnormal experience-dependent synapse development and maintenance. However, the biological function of MeCP2 and the pathogenic mechanisms of MeCP2 mutations remain unclear. Current evidence indicates that modulation of transcription is a major component of MeCP2 function. The genetic architecture of MeCP2-mediated gene regulation in the brain is complex, involving both cell autonomous and circuitry mechanisms, direct transcriptional targets and subsequent compensatory changes, and is dependent on developmental stage and postnatal experience. Therefore, characterizing altered gene expression profiles in relevant neuron types, brain regions, and developmental stages is essential for understanding the pathogenic mechanism of RTT. However, a major challenge in the analysis of gene expression in mammalian brain is the extraordinary cellular heterogeneity. We have developed a cell type-based Nucleic acid ImmunoPrecipitation technique (cNIP) to purify mRNA and miRNA from distinct cell types in the mouse brain. We have implemented cNIP by Cre/loxP-regulated expression of epitope-tagged polyA- binding protein (PABP, for mRNA) or argonaute2 (AGO2, for miRNA) in specific cell types. Here we propose to apply this technique to study mRNA and miRNA profiles in major classes of glutamatergic and GABAergic neurons in the motor and cognitive areas of the mouse neocortex at 3 stages of postnatal development. We will then examine the alterations in gene expression profiles in these neurons in germline MeCP2 mutant mice. We will further combine cell type-specific manipulation of MeCP2 and cell based gene expression analysis to distinguish the cell autonomous and non-autonomous action of MeCP2. Our cell based genomic approach will establish a new experimental paradigm to study the genetic architecture of MeCP2 mutations which can be applied to neurodevelopmental disorders in general. Simultaneous analysis of mRNA and miRNA profiles will generate a comprehensive molecular portrait of the relevant cell types and their developmental trajectories. Because of the increasingly well-defined role of these neuron populations in neural synchrony and network oscillations, the rich molecular information can be readily "plugged in" to the functional units of neural circuits, linking genetic mutations and altered gene networks to neural networks, brain dynamics, and cognitive dysfunction. PUBLIC HEALTH RELEVANCE: We will develop novel genetic and genomic technology and use mouse models to study the pathogenic mechanism of RTT syndrome, one of the best characterized of the autism spectrum neurodevelopmental disorders.